Method of extracting and method of purifying an effective substance

ABSTRACT

A method for extracting a hydrophobic group-containing water-soluble organic compound, comprising the step of bringing an aqueous solution containing the hydrophobic group-containing water-soluble organic compound and a saccharide into contact with a polar organic solvent to obtain an aqueous phase and an organic phase, whereby the hydrophobic group-containing water-soluble organic compound is transferred to the organic phase. The saccharide concentration of the aqueous solution may be at least 12 g per 100 ml of the aqueous solution. The aqueous solution may further contain a phase separation assisting agent. The phase separation assisting agent may be selected from the group consisting of sodium chloride, sodium citrate, magnesium sulfate, and ammonium sulfate.

TECHNICAL FIELD

[0001] The present invention relates to a method for extracting ahydrophobic group-containing water-soluble organic compound from anaqueous solution containing the hydrophobic group-containing:water-soluble organic compound (e.g., an extract derived from an animalor a plant as well as an enzyme reaction solution) with high purity andhigh yield.

BACKGROUND ART

[0002] As represented by crude drug extracts, a number of naturalcompounds having a variety of physiological activities are known. Thesenatural compounds are also called physiologically active substances. Anumber of physiologically active substances are generally purified byconducting extraction using a material containing the physiologicallyactive substance and water, warm water or low-concentration aqueousalcohol solution to obtain an extract solution, concentrating theextract solution, and subjecting the concentrated extract solution tocolumn chromatography. However, such a purifying method requires a largecolumn and equipment accompanying therewith in order to produce a largeamount of a physiologically active substance. A small column has verypoor efficiency. Therefore, a purified physiologically active substanceis very expensive.

[0003] Attempts have been made to purify physiologically activesubstances by a solvent extraction method. However, a method of addingan organic solvent which is inherently immiscible with water, such asethyl acetate, butanol, and chloroform, to an aqueous solution,stirring, allowing the solution to stand to obtain two phases, i.e.,aqueous phase and organic solvent phase, and recovering thephysiologically active substance transferred to the organic solventphase, cannot be used for foods due to safety problems. Even when aphysiologically active substance is used for applications other thanfoods, some physiologically active substances are inefficientlyextracted using an organic solvent since the physiologically activesubstances are not significantly transferred to the organic solventphase. Since some organic solvents which can be used for foods, such asethanol and acetone, are miscible with water, these organic solventscannot be used to extract and purify a physiologically active substancefrom aqueous solution.

[0004] Hesperidin is a representative flavonoid which is contained inorange juice. Flavonoids represented by hesperidin are known to havephysiological actions described below, for example. Hesperidin and rutinwere previously called vitamin P, and have long been known to have anaction that lowers blood pressure (Shintaro Kamiya, Shin-Vitamin-Gaku,[New Vitamin Study] (The Vitamin Society of Japan) 1969, p439). It hasalso been reported that hesperidin has the following physiologicalactions: anti-inflammatory action, analgesic action (E, M. Galati etal., Il Farmaco, 49, 709-712(1994)), antiallergic action (HideakiMatsuda et al.; Yakugaku Zasshi [Journal of Pharmacology], 111,193-198(1991), J. A. Da Silva Emim et al.; J. Pharm. Pharmacol., 46,118-712(1994)), an action that reduces LDL-cholesterol to ameliorateblood cholesterol levels (M. T. Monforte et al.; Il Farmaco,50,595-599(1995)), and carcinostatic action (T. Tanaka, et al.; CancerResearch, 54, 4653-4659(1994), T. Tanaka, et al.; Cancer Research, 57,246-252(1997), T. Tanaka, et al.: Carcinogenesis, 18, 761-769(1997), T.Tanaka, et al.: Carcinogenesis, 18, 957-965(1997)). Further, in recentstudies, it is expected that hesperidin also has an action that promotesdifferentiation of fat precursor cells to ameliorate conditions, such asdiabetes. Diosmin has a vigorous antioxidant activity.

[0005] A medical agent containing Diosmin and hesperidin is utilized asa therapeutic drug for venous insufficiency, hemorrhoids, and the like(C. Labrid: Angiology, 45, 524-530(1994)). It has also been reportedthat hesperidin alone, Diosmin alone, and a mixture of hesperidin andDiosmin suppresses oral cancer, esophageal cancer, colorectal cancer,and the like (T. Tanaka, et al.; Cancer Research, 54, 4653-4659(1994),T. Tanaka, et al.; Cancer Research, 57, 246-252(1997), T. Tanaka, etal.; Carcinogenesis, 18, 761-769(1997), T. Tanaka, et al.;Carcinogenesis, 18, 957-965(1997)).

[0006] Naringin and neohesperidin are known as bitter substances ofcitrus, and are used in foods and beverages for the purpose of providingbitterness.

[0007] Further, it has been recently revealed that isoflavoneeffectively improves bone density, suppresses occurrence of breastcancer, and the like (Toda et al. Foods and Ingredients Journal ofJapan, No. 172, 83-89 (1997)).

[0008] Hesperidin and rutin are inherently insoluble in acetone.

[0009] On the other hand, flavonoids, such as hesperidin, naringin,neohesperidin, and rutin, are poorly soluble in water. In order toovercome this drawback, i.e., the poor solubility, attempts have beenmade to efficiently solubilize these poorly soluble compounds. Forexample, a method of improving the solubility of flavonoids, such ashesperidin, naringin, neohesperidin, and rutin, by enzymaticglycosylation, is known (Japanese Laid-Open Publication No. 7-107972).

[0010] A method of improving the solubility of catechin, caffeic acid,kojic acid, hydroquinone, catechol, resorcinol, protocatechuic acid,gallic acid, vanillin, daidzein, genistein, α-resorcylic acid andphloroglucinol other than the above-described flavonoids by enzymaticglycosylation for the same purpose, is known (Japanese Publication forOpposition No. 7-36758 and T. Nishimura, J. Ferment. Bioeng., 78 (1994)p37).

[0011] However, since the water solubility of the glycoside itself isimproved, the glycoside cannot be efficiently extracted in a solventimmiscible with water. Also, due to safety problems, columnchromatography, such as adsorption chromatography, is required forpurification of glycosides from enzyme reaction solutions in whichglycosylation is conducted.

[0012] In conventional purification methods, in order to obtainpartially purified flavonoids, catechins, phenols and glycosides thereoffrom natural materials, a method of conducting extraction using thenatural material and alkaline aqueous solution, organic solvent, hotwater, or the like and then purifying the extract solution by columnchromatography, has been employed. However, extraction and purificationusing a safe organic solvent which can be used for foods are requiredfor obtaining a large amount of these substances with high yield andwith low cost. However, since acetone which can be utilized for foods ismiscible with water, it is not possible to extract these substancesusing acetone. Most physiologically active substances which areeffective in the food and drug fields have properties such that they areeasily soluble in a high polarity solvent, such as water, ethanol, andacetone, while they are poorly soluble in a low polarity solvent, andtherefore, cannot be efficiently extracted from a low polarity solvent.

DISCLOSURE OF THE INVENTION

[0013] As a result of diligent studies, the present inventors have foundthat even when using an organic solvent, which is inherently difficultto separate from aqueous phase where the organic solvent is mixed withwater or hot water, if a substance having water holding capacity, suchas a saccharides is present in an aqueous solution containing aphysiologically active substance, it is possible to easily separateaqueous phase from organic phase after mixing and stirring the aqueoussolution and the organic solvent. The present inventors also found thatthe physiologically active substance was transferred to the organicphase. Further, the present inventors found that by increasing the ionicstrength of the aqueous solution by adding salt (e.g., sodium chlorideand sodium citrate) or an organic acid thereto, an organic solvent whichis not otherwise separated from aqueous phase or which is otherwisedifficult to separate from aqueous phase even if a saccharide is presentcan be separated from aqueous phase, and a physiologically activesubstance can be efficiently transferred to the organic solvent phase.Based on these findings, the present inventors completed the presentinvention.

[0014] The method of the present invention is a method for extracting ahydrophobic group-containing water-soluble organic compound, comprisingthe step of bringing an aqueous solution containing the hydrophobicgroup-containing water-soluble organic compound and a saccharide intocontact with a polar organic solvent to obtain an aqueous phase and anorganic phase, whereby the hydrophobic group-containing water-solubleorganic compound is transferred to the organic phase.

[0015] In one embodiment, the saccharide concentration of the aqueoussolution may be at least 12 g per 100 ml of the aqueous solution.

[0016] In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be a water-soluble aromatic compound.

[0017] In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be selected from the group consisting of phenolderivatives and glycosides thereof.

[0018] In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be selected from the group consisting ofhydroquinone glycoside, catechin, salicin, hesperidin, hesperidinglycosides, caffeic acid, salicyl alcohol, and elladitannin.

[0019] In one embodiment, the aqueous solution may further contain aphase separation assisting agent.

[0020] In one embodiment, the phase separation assisting agent may be asalt or an organic acid.

[0021] In one embodiment, the phase separation assisting agent may beselected from the group consisting of sodium chloride, sodium citrate,magnesium sulfate, and ammonium sulfate.

[0022] In one embodiment, the polar organic solvent may betetrahydrofuran or acetonitrile.

[0023] In one embodiment, the polar organic solvent may betetrahydrofuran, acetonitrile, acetone, or isopropyl alcohol.

[0024] In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be derived from an enzyme reaction solution.

[0025] In one embodiment, the enzyme reaction solution may be aglycosylation reaction solution.

[0026] In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

[0027] In one embodiment, the hydrophobic group-containing water-solubleorganic compound may be derived from an organism selected from animalsor plants.

[0028] In one embodiment the hydrophobic group-containing water-solubleorganic compound may be derived from fruit juice.

[0029] In one embodiment, the aqueous solution may be prepared byconcentrating an enzyme reaction solution containing the hydrophobicgroup-containing water-soluble organic compound and the saccharide.

[0030] In one embodiment, the enzyme reaction solution may be aglycosylation reaction solution.

[0031] In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

[0032] In one embodiment, the aqueous solution may be prepared byconcentrating or diluting an extract of an organism, wherein the extractcontains the hydrophobic group-containing water-soluble organic compoundand the saccharide, and the organism is an animal or a plant.

[0033] In one embodiment, the aqueous solution may be prepared byconcentrating fruit juice.

[0034] In one embodiment, the aqueous solution may be prepared by addingthe phase separation assisting agent to an enzyme reaction solutioncontaining the hydrophobic group-containing water-soluble organiccompound and the saccharide, or a concentrate thereof.

[0035] In one embodiment, the enzyme reaction solution may be aglycosylation reaction solution.

[0036] In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

[0037] In one embodiment, the aqueous solution may be prepared by addingthe phase separation assisting agent to an extract of an organism, aconcentrate thereof, or a diluent thereof, wherein the extract containsthe hydrophobic group-containing water-soluble organic compound and thesaccharide, and the organism is an animal or a plant.

[0038] In one embodiment, the aqueous solution may be prepared by addingthe phase separation assisting agent to fruit juice or a concentratethereof.

[0039] The purifying method of the present invention is a method forpurifying a phenol derivative glycoside, comprising the steps ofbringing a first aqueous solution containing a phenol derivative, aphenol derivative glycoside and a saccharide into contact with a polarorganic solvent to obtain a first aqueous phase and an organic phasecontaining a small amount of water, whereby the phenol derivative andthe phenol derivative glycoside are transferred to the organic phase,recovering the organic phase containing the small amount of water,removing the polar organic solvent from the organic phase containing thesmall amount of water to obtain a second aqueous solution containing thephenol derivative and the phenol derivative glycoside, bringing thesecond aqueous solution into contact with ethyl acetate to obtain asecond aqueous phase and an ethyl acetate phase, whereby the phenolderivative is transferred to the ethyl acetate phase, recovering thesecond aqueous phase, and concentrating and cooling the second aqueousphase to precipitate the phenol derivative glycoside.

[0040] In one embodiment, the phenol derivative and the phenolderivative glycoside may be derived from a phenol derivativeglycosylation reaction solution.

[0041] In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

[0042] In one embodiment, the first aqueous solution may further containa phase separation assisting agent.

[0043] In one embodiment, the glycosylation reaction solution may be ahesperidin or hydroquinone glycosylation reaction solution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 is a graph showing an influence of glucose concentration ontetrahydrofuran extraction.

[0045]FIG. 2 is a graph showing the transfer rates of hydroquinoneglycoside and glucose to a tetrahydrofuran phase.

BEST MODE FOR CARRYING OUT THE INVENTION

[0046] Hereinafter, the present invention will be described in detail.As used herein, concentration is represented by grams per 100 cubiccentimeters of solution unless otherwise described. For example, “10%sodium chloride solution” refers to a sodium chloride solution in which10 g of sodium chloride is dissolved per 100 cubic centimeters ofsolution.

[0047] A method according to the present invention is a method ofextracting a hydrophobic group-containing water-soluble organiccompound. The method of the present invention comprises the step ofcontacting an aqueous solution containing a hydrophobic group-containingwater-soluble organic compound and a saccharide with a polar organicsolvent to obtain an aqueous phase and an organic phase so that thehydrophobic group-containing water-soluble organic compound istransferred to the organic phase.

[0048] (1) Hydrophobic Group-Containing Water-Soluble Organic Compound

[0049] As used herein, “hydrophobic group-containing water-solubleorganic compound” refers to an organic compound which contains ahydrophobic group and is soluble in water.

[0050] As used herein, “water-soluble” compound refers to a compoundthat at least 0.01 g can be dissolved in one liter of water at 20° C.Preferably, at least 0.1 g, more preferably at least 1 g, even morepreferably at least 5 g, and most preferably at least 10 g, of ahydrophobic group-containing water-soluble organic compound can bedissolved in one liter of water at 20° C. There is no particular upperlimit to the solubility, although the solubility is preferably no morethan 300 g in one liter of water at 20° C. More preferably, thesolubility is no more than 100 g in one liter of water at 20° C.

[0051] A hydrophobic group is preferably a hydrophobic group containingat least three carbon atoms, and more preferably an aromatic residue.Examples of hydrophobic group-containing water-soluble organic compoundsinclude flavonoids, isoflavones, phenolic compounds, flavonoidglycosides, isoflavone glycosides, phenolic compound glycosides,hydroquinone glycosides, anthracene glycosides, water-soluble aromaticcompounds (e.g., chalcone glycosides), terpene glycosides, steroidglycosides, triterpenoid glycosides, alkaloid glycosides, andC-glycosides. Preferably, the hydrophobic group-containing water-solubleorganic compound is a water-soluble aromatic compound.

[0052] As used herein, “water-soluble aromatic compound” refers to acompound which is soluble in water and has an aromatic group.

[0053] The water-soluble aromatic compound is preferably selected fromthe group consisting of phenol derivatives and glycosides thereof.

[0054] “Phenol derivative” refers to a compound having a phenol backbone(i.e., a benzene ring) or a flavonoid backbone and having a hydroxylgroup linked to the phenol backbone or the flavonoid backbone, includingphenol and kojic acid. Examples of the phenol derivative include acompound having a phenolic hydroxyl group on a single phenol orflavonoid backbone, and a compound having at least two phenolic hydroxylgroups on a single phenol or flavonoid backbone. Hereinafter, for thesake of convenience, compounds having one phenolic hydroxyl group on asingle phenol or flavonoid backbone are called monophenol typecompounds, and compounds having at least two phenolic hydroxyl groups ona single phenol or flavonoid backbone are called polyphenol typecompounds.

[0055] Compounds having two phenolic hydroxyl groups on a single phenolor flavonoid backbone are called diphenol compounds.

[0056] A phenol derivative glycoside having a phenolic hydroxyl group isalso included as a phenol derivative.

[0057] Examples of monophenol type compounds having one phenolichydroxyl group on a single phenol or flavonoid backbone include phenol,salicyl alcohol, kojic acid, dimethoxy phenol, acetaminophen, vanillin,and daidzein.

[0058] Examples of monophenol compounds also include monophenol typeflavonoid type compounds. Examples of monophenol type flavonoid typecompounds include monophenol type flavone type compounds, monophenoltype isoflavone type compounds, monophenol type flavonol type compounds,monophenol type flavanone type compounds, monophenol type flavanonoltype compounds, monophenol type catechin type compounds, monophenol typeaurone type compounds, monophenol type chalcone type compounds, andmonophenol type dihydrochalcone type compounds.

[0059] Examples of dimethoxyphenols include 2,3-dimethoxy phenol,2,4-dimethoxy phenol, 2,5-dimethoxy phenol, 2,6-dimethoxy phenol,3,4-dimethoxy phenol, and 3,5-dimethoxy phenol. 3,4-dimethoxy phenol and3,5-dimethoxy phenol are preferable.

[0060] Examples of polyphenol type compounds having at least twophenolic hydroxyl groups on a single phenol or flavonoid backboneinclude hydroquinone, hesperetin, epigallocatechin, epicatechin gallate,anthocyanidin type compounds, anthocyanin type compounds, caffeic acid,catechol, resorcinol, protocatechuic acid, gallic acid, genistein,β-resorcylic acid, and phloroglucinol.

[0061] Examples of diphenol compounds also include diphenol typeflavonoid type compounds. Examples of diphenol type flavonoid typecompounds include diphenol type flavone type compounds, diphenol typeisoflavone type compounds, diphenol type flavonol type compounds,diphenol type flavanone type compounds, diphenol type flavanonol typecompounds, diphenol type catechin type compounds, diphenol type auronetype compounds, diphenol type chalcone type compounds, and diphenol typedihydrochalcone type compounds.

[0062] Examples of resorcylic acids include α-resorcylic acid,β-resorcylic acid, and γ-resorcylic acid. In the present invention,β-resorcylic acid is preferable.

[0063] As used herein, a “phenol derivative glycoside” is a substance inwhich a phenol derivative moiety is linked to one or more saccharidemoiety with glycoside linkage(s). The phenol derivative glycoside may bea mono-glucopyranoside (e.g., hydroquinone-O-α-D-glucopyranoside,salicin, caffeic acid-O-α-D-glucopyranoside, 3,4-dimethoxyphenol-O-α-D-glucopyranoside, and catechin-O-α-D-glucopyranoside), adiglucopyranoside in which a saccharide moiety is additionally linked tothe above-described mono-glucopyranoside (e.g., a hesperidinderivative), a triglucopyranoside, and the like.

[0064] As used herein, a “glycoside” is a substance in which an aglyconis linked to one or more saccharide moieties with glycoside linkage(s).The polymerization degree of the saccharide moiety is preferably 1-10,more preferably 1-5, and even more preferably 1-3. The saccharide moietycan be a monosaccharide moiety or a disaccharide moiety. As used herein,glucosides are included in the definition of the glycoside. Glucosidesare glycosides in which one or more glucose moieties are linked to anaglycon.

[0065] The hydrophobic group-containing water-soluble organic compoundis preferably selected from the group consisting of salicin, coniferin,arbutin, sennoside, stevioside, rubsoside, rutin, hesperidin, naringin,daidzein, genistin, barbaroin, vanillin, saponins, berberine,kaempferol, baicalin, capillarin, catechin, corydaline, esculetin,epicatechin, gingerols, glycyrrhizin, Diosmin, neohesperidin, caffeicacid, salicyl alcohol, elladitannin, and hydroquinone. The hydrophobicgroup-containing water-soluble organic compound is more preferablyselected from the group consisting of hydroquinone glycoside, catechin,salicin, hesperidin, hesperidin glycosides, caffeic acid, salicylalcohol and elladitannin.

[0066] The hydrophobic group-containing water-soluble organic compoundmay be present in aqueous solution at any concentration. Theconcentration of the hydrophobic group-containing water-soluble organiccompound is preferably 0.01% to 50%, more preferably 0.1% to 40%, evenmore preferably 0.5% to 30%, still even more preferably 1% to 20%, andmost preferably 5% to 15%. If the concentration of the hydrophobicgroup-containing water-soluble organic compound present in aqueoussolution is excessively low, the purification efficiency may be poor. Ifthe concentration of the hydrophobic group-containing water-solubleorganic compound present in aqueous solution is excessively high, thehydrophobic group-containing water-soluble organic compound mayprecipitate. A concentration at which the hydrophobic group-containingwater-soluble organic compound does not precipitate is preferable.

[0067] The hydrophobic group-containing water-soluble organic compoundmay be derived from an enzyme reaction solution containing thehydrophobic group-containing water-soluble organic compound. As usedherein, an enzyme reaction solution refers to a solution obtained bysubjecting any starting material to an enzyme reaction. Examples of suchan enzyme reaction solution include a glycosylation reaction solution, ahydrolysis reaction solution, a transfer reaction, and a condensationreaction solution for the hydrophobic group-containing water-solubleorganic compound. The enzyme reaction solution preferably contains asaccharide. The enzyme reaction solution is typically a reactionsolution after a reaction has proceeded and a reaction product has beenproduced.

[0068] An example of a glycosylation reaction is representatively aglycosyl transfer reaction for a glycosyl transfer acceptor which iscatalyzed by cyclodextrin glucanotransferase. Examples of such aglycosyl transfer acceptor include a flavonoid containing a saccharidein the structure thereof, a flavonoid not containing a saccharide in thestructure thereof, a phenol compound, and a phenolic compound glycoside.Examples of a representative glycosyl transfer acceptor includehesperidin, naringin, neohesperidin, and rutin.

[0069] Another example of the glycosylation reaction is a glycosyltransfer reaction for a glycosyl transfer acceptor, 5 which is catalyzedby a transferring type amylase. Examples of such a glycosyl transferacceptor include catechin, caffeic acid, kojic acid, hydroquinone,catechol, resorcinol, protocatechuic acid, gallic acid, vanillin,daidzein, genistein, α-resorcylic acid and phloroglucinol.

[0070] The glycosylation reaction solution is preferably a glycosylationreaction solution for hesperidin or hydroquinone.

[0071] Examples of an enzyme catalyzing an enzyme reaction include, inaddition to cyclodextrin glucanotransferase and transferring typeamylase, α-amylase, pullulanase, amylomaltase, D-enzyme, neopullulanase,cyclodextrinase, α-glucosidase, cellulase, β-glucosidase, andβ-galactosidase.

[0072] The enzyme reaction solution containing the hydrophobicgroup-containing water-soluble organic compound may be designed andobtained by a method known to those skilled in the art.

[0073] The hydrophobic group-containing water-soluble organic compoundmay also be derived from any natural material containing the hydrophobicgroup-containing water-soluble organic compound. The hydrophobicgroup-containing water-soluble organic compound may be derived from, forexample, an organism (e.g., an animal or a plant). Alternatively, ananimal extract or a plant extract can be used. The animal extract refersto any substance extracted from an animal. The plant extract refers toany substance extracted from a plant. For example, any hydrophobicgroup-containing water-soluble organic compound obtained from a leaf,stem, root, flower, fruit, or the like of a plant can be used. Examplesof a plant material include soybean, processed soybean products,sea-cucumber, Chinese nutgall, Scutellaria root (Scutellariae Radix),aloe, Rehmannia root (Rehmanniae Radix), Asiatic ginseng (Panaxginseng), Peony root (Paeoniae Radix), Gardenia jasminoides, Glycyrrhiza(Glycyrrhizae Radix), Bupleurum root (Bupleuri Radix), Rhubarb (Rheirhizome), Houttuyniacordata, Cowberry (Vaccinium vitis-idaea), tea,Sweet tea (Chinese blackberry tea; Rubus suanissm S.Lee), and citrus(e.g., the fruit of orange). Any hydrophobic group-containingwater-soluble organic compound present in the body of an animal can beused.

[0074] (2) Saccharides

[0075] As used herein, saccharides refer to compounds having the generalformula Cn(H₂O)_(m). Saccharides are grouped into monosaccharides,oligosaccharides, and polysaccharides according to the number ofconstituents, i.e., saccharide units. In the present invention,monosaccharides and oligosaccharides are preferable. In the presentinvention, a saccharide, which is soluble in water or, if not, has waterholding capacity, is preferable.

[0076] Examples of monosaccharides include D-glucose, galactose,fructose, arabinose, xylose, and rhamnose. A preferable monosaccharideis D-glucose.

[0077] Oligosaccharides as used herein refer to substances obtained bydehydration-condensation of 2 to 10 monosaccharides. An oligosaccharidehas preferably 2 to 9 saccharide units, more preferably 2 to 8saccharide units, and even more preferably 2 to 7 saccharide units.Examples of oligosaccharide include sucrose, lactose,malto-oligosaccharides, galacto-oligosaccharides,lacto-oligosaccharides, and fructo-oligosaccharides. Examples of themalto-oligosaccharides include maltose, maltotriose, maltotetraose,maltopentaose, and maltohexaose, maltoheptaose, malto-octaose,maltononaose, and maltodecaose. An oligosaccharide may be astraight-chain oligosaccharide or a branched-chain oligosaccharide. Anoligosaccharide may have an intramolecular ring structure.

[0078] Polysaccharides as used herein refer to substances generated bydehydration-condensation of at least 11 monosaccharides. Apolysaccharide preferably has at least one α-1,4 linkage. Examples ofpolysaccharides include dextrin, amylose, amylopectin, starch, dextran,and cellulose.

[0079] Dextrins refer to substances obtained by lowering the molecularweight of starch by a chemical or enzymatic method. Examples of dextrinsinclude British gum, yellow dextrin, white dextrin, PINE-DEX (MatsutaniChemical Industry Co., Ltd.), SUNDECK (Sanwa Cornstarch Co., Ltd.), andTetrup (Hayashibara Shoji, Inc.).

[0080] Amylose is a straight-chain molecule composed of glucose unitslinked together by α-1,4 linkages. Amylose is contained in naturalstarch.

[0081] Amylopectin is a branched-chain molecule composed of glucoseunits linked together by α-1,4 linkages to which glucose units arelinked together by α-1,6 linkages. Amylopectin is contained in naturalstarch. As amylopectin, for example, waxy corn starch consisting of 100%amylopectin may be used.

[0082] Starch is a mixture of amylose and amylopectin. As starch, anystarch which is usually commercially available may be used. The ratio ofamylose to amylopectin contained in starch varies depending on the typeof plant which produces the starch. The majority of starch contained inwaxy rice, waxy corn, and the like is amylopectin. Starch is dividedinto natural starch, starch degradation products, and processed starch.

[0083] Natural starch is divided into tuber starch and cereal starchaccording to a raw material from which it is derived. Examples of tuberstarch include potato starch, tapioca starch, sweet potato starch, kudzustarch, bracken starch, and the like. Examples of cereal starch includecorn starch, wheat starch, rice starch, and the like.

[0084] Processed starch is starch obtained by subjecting natural starchto treatment, such as hydrolysis, esterification, gelatinization, or thelike, to confer properties for better ease of utilization. A widevariety of processed starches are available which have variouscombinations of properties, such as, for example, temperature at whichgelatinization starts, the viscosity of the starch paste, thetransparency of the starch paste, aging stability, and the like. Thereare various types of processed starch. An example of such starch isstarch which is obtained by immersing starch granules in acid at atemperature of no more than the gelatinization temperature of the starchso that starch molecules are cleaved but starch granules are not broken.

[0085] Starch degradation products are oligosaccharides orpolysaccharides obtained by subjecting starch to treatment, such asenzyme treatment, hydrolysis, or the like, which have a lower molecularweight than before the treatment. Examples of the starch degradationproducts include starch degraded by a debranching enzyme, starchdegraded by phosphorylase, and starch partially degraded by hydrolysis.

[0086] Starch degraded by a debranching enzyme is obtained by allowing adebranching enzyme to act on starch. By changing the action time of thedebranching enzyme to various extents, starch degraded by a debranchingenzyme in which branching portions (i.e., α-1,6-glucoside linkage) arecleaved to any extent can be obtained. Examples of the starch degradedby a debranching enzyme include degradation products of 4 to 10,000saccharide units having 1 to 20α-1,6-glucoside linkages, degradationproducts of 3 to 500 saccharide units without any α-1,6-glucosidelinkages, malto-oligosaccharide, and amylose. In the case of starchdegraded by a debranching enzyme, the distribution of the molecularweight of the resultant degradation products may vary depending on thetype of starch to be degraded. The starch degraded by a debranchingenzyme may be a mixture of saccharide chains having various lengths.

[0087] Dextrin and starch partially degraded by hydrolysis refer todegradation products obtained by degrading starch partially by theaction of an acid, an alkali, an enzyme, or the like. In the presentinvention, the number of saccharide units contained in dextrin andstarch partially degraded by hydrolysis is preferably about 10 to about100,000, more preferably about 50 to about 50,000, and even morepreferably about 100 to about 10,000. In the case of dextrin and starchpartially degraded by hydrolysis, the distribution of the molecularweight of the resultant degradation products may vary depending on thetype of starch to be degraded.

[0088] Dextrin and the starch partially degraded by hydrolysis may be amixture of saccharide chains having various lengths.

[0089] Dextran refers to α-1,6-glucan.

[0090] Cellulose is a straight-chain molecule composed of glucose unitslinked together by β-1,4-glucoside linkages.

[0091] The saccharide may be a single compound or a mixture of aplurality of compounds.

[0092] The saccharide may be originally contained in an aqueous solutioncontaining the hydrophobic group-containing water-soluble organiccompound, or may be added to an aqueous solution containing thehydrophobic group-containing water-soluble organic compound. Thesaccharide is preferably originally contained in an aqueous solutioncontaining the hydrophobic group-containing water-soluble organiccompound. Examples of such an aqueous solution include theabove-described glycosylation reaction solutions and fruit juice.

[0093] The saccharide preferably is a small molecule. When a solutioncontains a relatively high molecular weight polysaccharide, as does aglycosylation reaction solution, an enzyme cleaving saccharide chains,such as glucoamylase, may be added to the solution which is allowed toreact, thereby degrading the polysaccharide to monosaccharides oroligosaccharides. It is preferable that a polysaccharide in an aqueoussolution is degraded to monosaccharides or oligosaccharides before beingbrought into contact with an organic solvent. It is preferable that allsaccharides are dissolved in an aqueous solution. However, saccharidemay be partially suspended in an aqueous solution as long as thesaccharide does not interfere with separation of the aqueous solutionfrom an organic solvent.

[0094] The saccharide concentration of an aqueous solution may be anyconcentration. The saccharide concentration of an aqueous solution ispreferably at least 12%, more preferably at least 20%, even morepreferably at least 30%, still even more preferably at least 40%, andmost preferably at least 50%. The upper limit of the saccharideconcentration is any concentration as long as the saccharide and thehydrophobic group-containing water-soluble organic compound do notprecipitate. For example, the upper limit is no more than 90%, no morethan 80%, no more than 70%, no more than 60%, or no more than 55%.

[0095] The saccharide concentration of an aqueous solution may bemeasured by a method known in the art. For example, as a simple method,there is a measuring method using a Brix scale. The measuring methodusing a Brix scale is simple, but cannot measure each of saccharideseparately. In order to measure each of the saccharides separately, forexample, an aqueous solution containing saccharides may be subjected toHPLC using a column LiChrosorb NH₂ (manufactured by Merck; 4.0×250 mm)and using a mixture of water and acetonitrile at 25:75 (v/v) as a mobilephase, and the eluate may be measured using an RI detector.

[0096] The pH of an aqueous solution is preferably 2 to 11, morepreferably 3 to 9, and even more preferably 4 to 8.

[0097] (3) Polar Organic Solvent

[0098] As used herein, “polar organic solvent” refers to an organicsolvent which has a solvent strength (ε⁰) to alumina of at least 0.4 andis miscible with distilled water. The solvent strength of a polarorganic solvent is preferably 0.42 to 0.98, more preferably 0.44 to0.95, and most preferably 0.44 to 0.90. A polar organic solvent may bean organic solvent having a permittivity of at least 7.0 at 20° C. Apolar organic solvent preferably has a permittivity of 7.3 to 40.0, morepreferably 7.4 to 39.0, and most preferably 7.5 to 38.0 at 20° C.

[0099] Examples of the solvent strength to alumina are shown in Table 1.Examples of the permittivity are shown in Table 2. Examples of polarorganic solvents used in the present invention include polar organicsolvents shown in the following Tables 1 and 2, which have a solventstrength or a permittivity within the above-described ranges. TABLE 1Solvent strength Solvent to alumina (ε⁰) fluoroalkane −0.25   n-pentane0.00 isooctane 0.01 hexane 0.01 n-decane 0.04 cyclohexane 0.04cyclopentane 0.05 carbon disulfide 0.15 carbon tetrachloride 0.18 xylene0.26 isopropyl ether 0.28 toluene 0.29 benzene 0.32 ethyl ether 0.38chloroform 0.40 methylene chloride 0.42 methyl isobutyl ketone 0.43tetrahydrofuran 0.45 ethylene dichloride 0.49 methyl ethyl ketone 0.51acetone 0.56 dioxane 0.56 ethyl acetate 0.58 methyl acetate 0.60dimethyl suffixed 0.62 aniline 0.62 diethyl amine 0.63 nitromethane 0.64acetonitrile 0.65 pyridine 0.71 isopropanol 0.82 n-propanol 0.82 ethanol0.88 methanol 0.95 ethylene glycol 1.11 acetic acid great water great

[0100] TABLE 2 Solvent e isooctane 1.94 n-hexane 1.88 n-heptane 1.92diethyl ether 4.33 cyclohexane 2.02 ethyl acetate 6.02 toluene 2.38chloroform 4.81 tetrahydrofuran 7.58 benzene 2.27 acetone 20.7dichloromethane 8.93 dioxane 2.25 propanol 20.33 ethanol 25.8dimethylformamide 36.7 acetonitrile 37.5 acetic acid 6.3 dimethylsulfoxide 4.7 methanol 32.7 water 81.1

[0101] A polar organic solvent is preferably tetrahydrofuran,isopropanol, acetonitrile, acetone, ethanol, methanol, propanol,pyridine, or dimethoxy sulfoxide, more preferably tetrahydrofuran,isopropanol, acetonitrile or acetone, and most preferablytetrahydrofuran or acetonitrile. When an aqueous solution furthercontains a phase separation assisting agent, a polar organic solvent ispreferably tetrahydrofuran, acetonitrile, acetone or isopropyl alcohol,and more preferably acetone or isopropyl alcohol.

[0102] A polar organic solvent is preferably a single compound. However,a mixture of at least two polar organic solvents may be used as long asan organic phase is not separated into two phases. A polar organicsolvent, which is appropriate for extraction of a hydrophobicgroup-containing water-soluble organic compound from an aqueoussolution, may be selected by those skilled in the art as required.

[0103] The amount of a polar organic solvent which is brought intocontact with an aqueous solution is representatively 0.1 to 10 times,and more preferably 0.2 to 2 times the volume of an aqueous solution.

[0104] (4) Phase Separation Assisting Agent

[0105] An aqueous solution may contain a phase separation assistingagent. As used herein, “phase separation assisting agent” refers to asubstance which assists separation of a mixture of an aqueous solutionand a polar organic solvent into an aqueous phase and an organic phase.Note that a saccharide and a hydrophobic group-containing water-solubleorganic compound are not phase separation assisting agents, even if theyhave an action of assisting phase separation. A phase separationassisting agent may be a salt having a salting-out effect and awater-soluble substance capable of enhancing ionic strength. A phaseseparation assisting agent may be a salt or an organic acid. Examples ofphase separation assisting agents include, but are not limited to,sulfates (e.g., ammonium sulfate and magnesium sulfate), sodium salts(e.g., sodium chloride and sodium sulfite), phosphates (e.g., potassiumphosphate, sodium phosphate, magnesium phosphate, and ammoniumphosphate), acetates (e.g., sodium acetate and potassium acetate),lactates (e.g., sodium lactate and magnesium lactate), organic acids(e.g., citric acid, sodium citrate, ascorbic acid, sodium ascorbate, andmalic acid), and ammonium chloride. A phase separation assisting agentis preferably a salt and is more preferably selected from the groupconsisting of sodium chloride, sodium citrate, magnesium sulfate andammonium sulfate.

[0106] A phase separation assisting agent may be contained in an aqueoussolution in a sufficient amount to assist phase separation. Such anamount is known to those skilled in the art. The concentration of aphase separation assisting agent contained in an aqueous solution ispreferably at least 5%, more preferably 10%, even more preferably atleast 15%, and most preferably at least 20%. There is no particularupper limit to the amount of a phase separation assisting agent,although it is preferably no more than 50% and more preferably no morethan 40%.

[0107] It is preferable that a phase separation assisting agent iscontained in an aqueous solution in advance. However, a phase separationassisting agent can be added while an aqueous solution and a polarorganic solvent are brought into contact with each other.

[0108] (Preparation of an Aqueous Solution Containing a HydrophobicGroup-Containing Water-Soluble Organic Compound and a Saccharide)

[0109] An aqueous solution containing a hydrophobic group-containingwater-soluble organic compound and a saccharide may be prepared by amethod known to those skilled in the art. Such an aqueous solution maybe an enzyme reaction solution which is not subjected to any treatmentafter the enzyme reaction has proceeded, or an enzyme reaction solutionwhich is concentrated, diluted, filtered, or pH-adjusted after theenzyme reaction has proceeded. Particularly, when the viscosity of anenzyme reaction solution is too high to be stirred, it is preferable todilute the enzyme reaction solution. Alternatively, an aqueous solutionmay be prepared by adding a phase separation assisting agent to anenzyme reaction solution containing a hydrophobic group-containingwater-soluble organic compound and a saccharide, or a concentratedsolution thereof.

[0110] An aqueous solution may also be an extract of an organismselected from animals or plants, which contains a hydrophobicgroup-containing water-soluble organic compound. Such an extract may beprepared by extracting an animal material or a plant material containinga hydrophobic group-containing water-soluble organic compound by amethod known in the art. An exemplary extraction method comprises:providing an animal material or a plant material containing ahydrophobic group-containing water-soluble organic compound into anextraction solvent, such as water (e.g., water having a temperature ofmore than 0° C. and less than 40° C.), warm water (e.g., water having atemperature of no less than 40° C. and less than 60° C.), hot water(e.g., water having a temperature of no less than 60° C. and less than100° C.), alcohol, pyridine, ethyl acetate, or a mixture thereof;allowing the hydrophobic group-containing water-soluble organic compoundto be transferred from the animal material or the plant material to theextraction solvent; removing the animal material and the plant materialfrom the extraction solvent to obtain an extract solution; andconcentrating or drying the extract solution if necessary. It ispreferable that the extract does not contain an organic solvent. Whenthe extraction solvent is an organic solvent, it is preferable to removethe organic solvent by concentrating the extract solution. The extractmay be liquid or solid. Juice obtained by squeezing an animal materialor a plant material is also herein included in the definition of anextract. An aqueous solution is preferably fruit juice. An animalmaterial may be the entire animal or any organ or tissue of an animal. Aplant material may be the entire plant or any organ (e.g., flower,fruit, seed, root, stem, and leaf) or tissue of a plant. An animalmaterial and a plant material to be subjected to extraction may beeither raw or dried. If an extract is an aqueous solution, the extractmay be used as it is in the present invention. An aqueous solution maybe prepared from an extract by concentration, dilution, or the like.Particularly, when the viscosity of an extract is too high to bestirred, it is preferable to dilute the extract. Note that as long as ahydrophobic group-containing water-soluble organic compound of interestis dissolved in an aqueous solution to be used in the method of thepresent invention, any other ingredient may be suspended therein.Alternatively, an aqueous solution may be prepared by adding a phaseseparation assisting agent to an animal extract containing a hydrophobicgroup-containing water-soluble organic compound and a saccharide, aplant extract containing a hydrophobic group-containing water-solubleorganic compound and a saccharide, or a concentrate or diluent of theseextracts. For example, an aqueous solution may be prepared by adding aphase separation assisting agent to fruit juice or a concentratethereof.

[0111] An effective ingredient selected from the group consisting ofsalicin, coniferin, arbutin, sennoside, stevioside, rubsoside, rutin,hesperidin, naringin, daidzein, genistin, barbaroin, vanillin, saponins,berberine, kaempferol, baicalin, capillarin, catechin, corydaline,esculetin, epicatechin, gingerols and glycyrrhizin, is preferablydissolved in an extract solution.

[0112] (Extraction of a Hydrophobic Group-Containing Water-SolubleOrganic Compound)

[0113] In a method according to the present invention, an aqueoussolution containing a hydrophobic group-containing water-soluble organiccompound and a saccharide is brought into contact with a polar organicsolvent to obtain an aqueous phase and an organic phase, thereby thehydrophobic group-containing water-soluble organic compound is allowedto be transferred to the organic phase.

[0114] An aqueous solution and a polar organic solvent may be broughtinto contact with each other by, for example, mixing the aqueoussolution and the polar organic solvent. Bringing an aqueous solution anda polar organic solvent into contact with each other is also calledextraction. A temperature at which an aqueous solution and a polarorganic solvent are brought into contact with each other is preferably10° C. to 50° C., more preferably 25° C. to 45° C., even more preferably20° C. to 40° C., and most preferably 25° C. to 35° C.

[0115] An aqueous solution and a polar organic solvent are mixed andstirred, followed by allowing them to stand, resulting in separation toan aqueous phase and an organic phase. In general, the aqueous phase andthe organic phase form respective layers, i.e., a water layer and anorganic layer. In general, the solvent layer, which has a greaterspecific gravity, is the lower layer. When an organic solvent which hasa smaller specific gravity than that of water is used, the water layertypically is the lower layer while the upper layer is the organic layer.

[0116] Typically, an aqueous phase contains a small amount of polarorganic solvent, while an organic phase contains a small amount ofwater. For example, acetone is added to an aqueous solution or asuspension containing a hydrophobic group-containing water-solubleorganic compound (e.g., hesperidin and rutin), which are in turn stirredand then allowed to stand. When they are separated into an aqueous phaseand an organic phase (acetone phase), a small amount of water isdissolved in the acetone phase. Therefore, compared with the case whenwater is not contained in the organic phase, the solubility of thehydrophobic group-containing water-soluble organic compound increases,the hydrophobic group-containing water-soluble organic compound isefficiently dissolved in acetone.

[0117] When an aqueous solution and a polar organic solvent are mixed tocontact each other, the mixture is preferably stirred. Examples of amethod for stirring include, but are not limited to, rotating, shaking,and both. In order to efficiently extract and purify a hydrophobicgroup-containing water-soluble organic compound, a multistagecounterflow partition apparatus (also called a continuous liquid-liquidextraction apparatus) can be used.

[0118] (Purification of a Phenol Derivative Glycoside)

[0119] The method of the present invention is particularly useful forpurification of a phenol derivative glycoside. Purification of a phenolderivative glycoside will be described as an example in more detail.

[0120] A phenol derivative glycoside may be formed by, for example,causing a saccharide (e.g., a malto-oligosaccharide or starch) to reactwith a phenol derivative in the presence of an enzyme. Typically, thisreaction reaches equilibrium at a certain level and does not proceedfurther. Therefore, in this enzyme reaction solution, the phenolderivative, the phenol derivative glycoside and the saccharide arepresent. When the enzyme reaction solution contains a polysaccharide oran oligosaccharide, a glycolytic enzyme, such as glucoamylase, may beadded to the enzyme reaction solution, followed by incubation, todegrade the polysaccharide or the oligosaccharide in the enzyme reactionsolution to monosaccharides. The degradation of the polysaccharide orthe oligosaccharide to monosaccharides increases the mole value, i.e.,molar concentration, without changing the total weight of thesaccharides, resulting in promotion of separation of an aqueous phaseand an organic phase. When the enzyme reaction solution and the polarorganic solvent are brought into contact with each other to obtain afirst aqueous phase and an organic phase containing a small amount ofwater, the phenol derivative and the phenol derivative glycoside aretransferred to the organic phase. As described above, in order topromote phase separation, a phase separation assisting agent may beadded to the aqueous phase to obtain an aqueous solution containing thephase separation assisting agent, and then the aqueous solution may bebrought into contact with the polar organic solvent.

[0121] Thereafter, the organic phase containing a small amount of wateris recovered. A phenol derivative and a phenol derivative glycosidecontain a hydrophobic portion which causes them to have a higheraffinity to an organic phase than that of a saccharide. Therefore, thephenol derivative and the phenol derivative glycoside are efficientlytransferred to the organic phase, the saccharide is less transferred tothe organic phase. Therefore, the phenol derivative and the phenolderivative glycoside are extracted in the recovered organic phase. Ingeneral, assuming that the same total amount of a polar organic solventis used for partition extraction, if the polar organic solvent isdivided into some aliquots and the aliquots are used to performextraction from an aqueous solution a plurality of times, the efficiencyof the extraction is greater than when the whole amount of the polarorganic solvent is used a single time to contact the aqueous solutionfor extraction. Therefore, a step of bringing an aqueous phase remainingafter an organic phase is recovered into contact with a polar organicsolvent to obtain an aqueous phase and an organic phase containing asmall amount of water again and recovering the organic phase, may beperformed twice or more. The recovery operation of an organic phase isperformed at least twice, the obtained organic phases may be combinedtogether and may be used in a subsequent step.

[0122] Thereafter, the polar organic solvent is removed from the organicphase containing a small amount of water. A method for removing thepolar organic solvent from the organic phase may be any method known tothose skilled in the art. Examples of such a method includeconcentration using an evaporator and evapor. The polar organic solventmay be completely removed, or may remain in a small amount as long as itdoes not interfere with a subsequent step. After the removal of thepolar organic solvent, the phenol derivative and the phenol derivativeglycoside remain in a small amount of water which has been contained inthe polar organic solvent. In this removal step, it is preferable toavoid much water from being removed. In this removal step, it ispreferable that the phenol derivative and the phenol derivativeglycoside do not precipitate. If the phenol derivative and the phenolderivative glycoside precipitate, it is preferable that the phenolderivative and the phenol derivative glycoside are dissolved by additionof water. Thus, a second aqueous solution containing the phenolderivative and the phenol derivative glycoside is obtained.

[0123] Thereafter, the second aqueous solution is brought into contactwith ethyl acetate to obtain a second aqueous phase and an ethyl acetatephase, so that the phenol derivative is transferred to the ethyl acetatephase.

[0124] Thereafter, the second aqueous phase is recovered. Since thephenol derivative does not contain a glycoside moiety, the phenolderivative has a higher affinity to the ethyl acetate phase than that ofthe phenol derivative glycoside. Therefore, the phenol derivative isefficiently transferred to the ethyl acetate phase, while the phenolderivative glycoside is less transferred to the ethyl acetate phase.Therefore, the phenol derivative glycoside remains in the recoveredaqueous phase. When a small amount of saccharide remains in the secondsolution, the saccharide remains in the aqueous phase. As in the step ofcontacting the aqueous solution and the polar organic solvent and thestep of recovering the organic phase, a step of bringing the aqueousphase remaining after the removal of the ethyl acetate phase intocontact with ethyl acetate to obtain an aqueous phase and an ethylacetate phase again and recovering the second aqueous phase may beperformed at least twice.

[0125] Note that although a method is herein described in which the stepof contacting an organic phase with ethyl acetate is performed after thestep of contacting the aqueous solution and the polar organic solventand the step of recovering the organic phase, the aqueous solution maybe brought into contact with ethyl acetate and an aqueous phase isrecovered before the aqueous phase is brought into contact with a polarorganic solvent.

[0126] Thereafter, the second aqueous phase is concentrated and cooledso as to precipitate the phenol derivative glycoside. It is preferableto concentrate the second aqueous phase so that the phenol derivativeglycoside in the second aqueous phase reaches a concentration of atleast 10%, preferably at least 15%, more preferably at least 20%, andmost preferably at least 25%. This is done by utilizing the fact thatthe saturation concentration in water of a phenol derivative glycosideis lower than the saturation concentration in water of a saccharide. Forexample, assuming that the saccharide is glucose and the phenolderivative glycoside is hydroquinone glycoside, the fact that thesaturation concentration in water of glucose is about 18% and thesaturation concentration in water of hydroquinone glycoside is about 10%is utilized.

EXAMPLES

[0127] Next, the present invention will be described in more detail byway of examples, although the present invention is not limited to theexamples.

Experimental Example 1 Influence of a Saccharide on Phase Separation

[0128] Glucose, sucrose and fructose were used as representatives ofrespective types of saccharide so that an influence thereof on phaseseparation of an aqueous solution and a polar organic solvent could bestudied.

[0129] Specifically, glucose, sucrose or fructose was dissolved in waterto prepare 10%, 15%, 20%, 25%, 30% and 50% aqueous solutions. 10 ml ofacetonitrile or tetrahydrofuran was added to 10 ml of each aqueoussaccharide solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Both acetonitrile and tetrahydrofuran are polarorganic solvents which are water miscible and when they are mixed withwater, the mixture does not undergo separation into an aqueous phase andan organic phase. After the stirring, the mixture was allowed to standfor 30 minutes, observing whether or not the mixture was separated intotwo phases, an aqueous phase and an organic phase. The results from theaddition of tetrahydrofuran or acetonitrile to each aqueous solution areshown in Tables 3 to 5. In the tables, THF represents tetrahydrofuranand AcCN represents acetonitrile. TABLE 3 Glucose 50% 30% 25% 20% 15%10% AcCN Yes Yes Yes Yes Yes No THF Yes Yes Yes No No No

[0130] TABLE 4 Sucrose 50% 30% 25% 20% 15% 10% AcCN Yes Yes Yes Yes YesNo THF Yes No No No No No

[0131] TABLE 5 Fructose 50% 30% 25% 20% 15% 10% AcCN Yes Yes Yes Yes YesNo THF Yes Yes No No No No

[0132] As shown in Tables 3 to 5, it was found that when acetonitrile ortetrahydrofuran was mixed with the aqueous saccharide solution, when acertain amount of saccharide was present, phase separation occurred.

[0133] According to the results, it was found that adjustment of thetype and concentration of a saccharide contained in an aqueous solutionwould facilitate phase separation and, for example, it is preferable toadjust a saccharide concentration of at least about 12%.

Experimental Example 2 Influence of a Salt on Phase Separation

[0134] Next, an influence of a salt on phase separation was studied.

[0135] Specifically, sodium chloride was added to an aqueous glucosesolution having a glucose concentration of 25% or 50% to a concentrationof 20% or 10%, respectively, to prepare a sodium chloride-containingaqueous glucose solution. 10 ml of acetone or isopropanol, which is apolar organic solvent, was added to 10 ml of each aqueous solution,followed by vigorous stirring using a separating funnel 5 minutes. Afterstirring, the mixture was allowed to stand for 30 minutes, observingwhether the mixture was separated into two phases. The results are shownin Table 6. TABLE 6 Glucose 50% + Glucose 25% + NaCl 20% NaCl 10%Acetone Yes No 2-ProOH Yes Yes

[0136] Sodium citrate was added to an aqueous solution having a dextrinconcentration of 25%, to a concentration of 20% to prepare a sodiumcitrate-containing aqueous dextrin solution. Note that dextrin is aPINE-DEX #1 manufactured by Matsutani Chemical Industry Co., Ltd., whichhas DE of 7 to 9. As used herein, “DE” is an index which shows thedegree of degradation of a starch and is a percentage of direct reducingsaccharide converted to glucose in the solid content. Magnesium sulfatewas added to an aqueous solution having a glucose concentration of 25%to a concentration of 10% to prepare an aqueous magnesiumsulfate-containing glucose solution. 10 ml of acetone, which is a polarorganic solvent, was added to 10 ml of each aqueous solution, followedby vigorous stirring using a separating agent for 5 minutes. Afterstirring, the mixture was allowed to stand for 30 minutes, observingwhether the mixture was separated into two phases. The results are shownin Table 7. TABLE 7 Dextrin 25% + Glucose 25% + Sodium Magnesium Citrate20% Sulfate 10% Acetone Yes Yes

[0137] As shown in Tables 6 and 7, when the sodium chloride-containingaqueous glucose solution, the sodium citrate-containing aqueous dextrinsolution and the magnesium sulfate-containing aqueous glucose solutionwere respectively mixed with acetone or isopropanol, phase separationoccurred. The addition of a salt, such as sodium chloride, sodiumcitrate, or magnesium sulfate, could lead to the occurrence of phaseseparation in a polar organic solvent which does not otherwise undergophase separation in the case of 50% aqueous saccharide solution. As aresult, it was found that a salt, such as sodium chloride, acts as aphase separation assisting agent. It is believed that a salt acts as aphase separation assisting agent by increasing the ionic strength of anaqueous solution in which the salt is dissolved. Therefore, anysubstance which can increase ionic strength is considered to be able tobe used as a phase separation assisting agent.

Example 1 Extraction of Catechins Example 1a

[0138] 1 g of a mixture of catechins (SUNPHENON®; manufactured by TaiyoKagaku Co., Ltd.) was dissolved in 100 ml of an aqueous solutioncontaining 30% glucose to obtain a sample aqueous solution. Theabsorbance at 280 nm of the sample aqueous solution was measured. 10 mlof tetrahydrofuran was added to 10 ml of the sample aqueous solution,followed by vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of the catechins extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 94.1% of the starting amount of catechinswas extracted in the tetrahydrofuran phase.

Example 1b

[0139] This additional extraction operation to Example 1a was conductedexcept for use of 10 ml of acetonitrile instead of 10 ml oftetrahydrofuran. As a result, it was found that by a single extractionoperation, 84.4% of the starting amount of catechins was extracted inthe acetonitrile phase.

Example 2 Extraction of Catechins Example 2a

[0140] 1 g of a mixture of catechins (SUNPHENON®; manufactured by TaiyoKagaku) was dissolved in 100 ml of an aqueous solution containing 30%glucose and 10% sodium chloride to obtain a sample aqueous solution. Theabsorbance at 280 nm of the sample aqueous solution was measured. 10 mlof acetone was added to 10 ml of the sample aqueous solution, followedby vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of the catechins extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 91.8% of the starting amount of catechinswas extracted in the acetone phase.

Example 2b

[0141] This additional extraction operation to Example 2a was conductedexcept for use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 93.2% of thestarting material of catechins was extracted in the isopropanol phase.

Example 3 Extraction of Hesperidin Glycosides) Example 3a

[0142] 1 g of hesperidin glycosides (manufactured by Toyo Sugar RefiningCo., Ltd.) were dissolved in 100 ml of an aqueous solution containing30% glucose to obtain a sample aqueous solution. The absorbance at 280nm of the sample aqueous solution was measured. 10 ml of tetrahydrofuranwas added to 10 ml of the sample aqueous solution, followed by vigorousstirring using a separating funnel for 5 minutes. Thereafter, themixture was allowed to stand for 30 minutes. As a result, the mixturewas separated into two phases. The lower phase (aqueous phase) wasrecovered, and the volume and the absorbance at 280 nm thereof weremeasured. The amount of hesperidin glycosides extracted in the upperphase (polar organic solvent phase) was calculated from a reduction inthe absorbance at 280 nm of the lower phase and the solution volume ofthe lower phase. As a result, it was found that by a single extractionoperation, 50.0% of the starting amount of hesperidin glycosides wasextracted in the tetrahydrofuran phase. Further, similar operations andmeasurements were performed twice more by adding 5 ml of tetrahydrofuranto the aqueous phase recovered after the tetrahydrofuran extraction. Asa result, it was found that a total of 80.0% of the hesperidinglycosides was extracted.

Example 3b

[0143] This additional extraction operation to Example 3a was conductedexcept for use of 10 ml of acetonitrile instead of 10 ml oftetrahydrofuran. As a result, it was found that by a single extractionoperation, 27.6% of the starting amount of hesperidin glycosides wasextracted in the acetonitrile phase.

Example 4 Extraction of Hesperidin Glycosides Example 4a

[0144] 1 g of hesperidin glycosides (Toyo Sugar Refining Co., Ltd.) weredissolved in 100 ml of an aqueous solution containing 30% glucose and10% sodium chloride to obtain a sample aqueous solution. The absorbanceat 280 nm of the sample aqueous solution was measured. 10 ml of acetonewas added to 10 ml of the sample aqueous solution, followed by vigorousstirring using a separating funnel for 5 minutes. Thereafter, themixture was allowed to stand for 30 minutes. As a result, the mixturewas separated into two phases. The lower phase (aqueous phase) wasrecovered, and the volume and the absorbance at 280 nm thereof weremeasured. The amount of hesperidin glycosides extracted in the upperphase (polar organic solvent phase) was calculated from a reduction inthe absorbance at 280 nm of the lower phase and the solution volume ofthe lower phase. As a result, it was found that by a single extractionoperation, 43.5% of the starting amount of hesperidin glycosides wasextracted in the acetone phase. Further, similar operations andmeasurements were performed twice more by adding 5 ml of acetone to theaqueous phase recovered after the acetone extraction. As a result, itwas found that a total of 90.0% of the hesperidin glycosides wasextracted.

Example 4b

[0145] This additional extraction operation to Example 4a was conductedexcept for use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 32.0% of thestarting amount of hesperidin glycosides was extracted in theisopropanol phase.

Example 5 Extraction of Salicin Example 5a

[0146] 1 g of salicin was dissolved in 100 ml of an aqueous solutioncontaining 30% glucose to obtain a sample aqueous solution. Theabsorbance at 280 nm of the sample aqueous solution was measured. 10 mlof tetrahydrofuran was added to 10 ml of the sample aqueous solution,followed by vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of salicin extracted in the upperphase (polar organic solvent phase) was calculated from a reduction inthe absorbance at 280 nm of the lower phase and the solution volume ofthe lower phase. As a result, it was found that by a single extractionoperation, 96.5% of the starting amount of salicin was extracted in thetetrahydrofuran phase.

Example 5b

[0147] This additional extraction operation to Example 5a was conductedexcept for use of 10 ml of acetonitrile instead of 10 ml oftetrahydrofuran. As a result, it was found that by a single extractionoperation, 98.1% of the starting amount of salicin was extracted in theacetonitrile phase.

Example 6 Extraction of Salicin Example 6a

[0148] 1 g of salicin was dissolved in 100 ml of an aqueous solutioncontaining 30% glucose and 10% sodium chloride to obtain a sampleaqueous solution. The absorbance at 280 nm of the sample aqueoussolution was measured. 10 ml of acetone was added to 10 ml of the sampleaqueous solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Thereafter, the mixture was allowed to stand for30 minutes. As a result, the mixture was separated into two phases. Thelower phase (aqueous phase) was recovered, and the volume and theabsorbance at 280 nm thereof were measured. The amount of salicinextracted in the upper phase (polar organic solvent phase) wascalculated from a reduction in the absorbance at 280 nm of the lowerphase and the solution volume of the lower phase. As a result, it wasfound that by a single extraction operation, 58.0% of the startingamount of salicin was extracted in the acetone phase.

Example 6b

[0149] This additional extraction operation to Example 6a was conductedexcept for use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 97.0% of thestarting amount of salicin was extracted in the isopropanol phase.

Example 7 Extraction of Caffeic Acid Example 7a

[0150] 1 g of caffeic acid was dissolved in 100 ml of an aqueoussolution containing 30% glucose to obtain a sample aqueous solution. Theabsorbance at 280 nm of the sample aqueous solution was measured. 10 mlof tetrahydrofuran was added to 10 ml of the sample aqueous solution,followed by vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of caffeic acid extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 88.6% of the starting amount of caffeicacid was extracted in the tetrahydrofuran phase.

Example 7b

[0151] This additional extraction operation to Example 7a was conductedexcept for use of 10 ml of acetonitrile instead of 10 ml oftetrahydrofuran. As a result, it was found that by a single extractionoperation, 76.2% of the starting amount of caffeic acid was extracted inthe acetonitrile phase.

Example 8 Extraction of Caffeic Acid Example 8a

[0152] 1 g of caffeic acid was dissolved in 100 ml of an aqueoussolution containing 30% glucose and 10% sodium chloride to obtain asample aqueous solution. The absorbance at 280 nm of the sample aqueoussolution was measured. 10 ml of acetone was added to 10 ml of the sampleaqueous solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Thereafter, the mixture was allowed to stand for30 minutes. As a result, the mixture was separated into two phases. Thelower phase (aqueous phase) was recovered, and the volume and theabsorbance at 280 nm thereof were measured. The amount of caffeic acidextracted in the upper phase (polar organic solvent phase) wascalculated from a reduction in the absorbance at 280 nm of the lowerphase and the solution volume of the lower phase. As a result, it wasfound that by a single extraction operation, 78.2% of the startingamount of caffeic acid was extracted in the acetone phase.

Example 8b

[0153] This additional extraction operation to Example 8a was conductedexcept for use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 87.2% of thestarting amount of caffeic acid was extracted in the isopropanol phase.

Example 9 Extraction of Salicyl Alcohol Example 9a

[0154] 1 g of salicyl alcohol was dissolved in 100 ml of an aqueoussolution containing 30% glucose to obtain a sample aqueous solution. Theabsorbance at 280 nm of the sample aqueous solution was measured. 10 mlof tetrahydrofuran was added to 10 ml of the sample aqueous solution,followed by vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The lower phase(aqueous phase) was recovered, and the volume and the absorbance at 280nm thereof were measured. The amount of salicyl alcohol extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 98.7% of the starting material of salicylalcohol was extracted in the tetrahydrofuran phase.

Example 9b

[0155] This additional extraction operation to Example 9a was conductedexcept for use of 10 ml of acetonitrile instead of 10 ml oftetrahydrofuran. As a result, it was found that by a single extractionoperation, 98.2% of the starting amount of salicyl alcohol was extractedin the acetonitrile phase.

Example 10 Extraction of Salicyl Alcohol Example b1a

[0156] 0.1 g of salicyl alcohol was dissolved in 100 ml of an aqueoussolution containing 30% glucose and 10% sodium chloride to obtain asample aqueous solution. The absorbance at 280 nm of the sample aqueoussolution was measured. 10 ml of acetone was added to 10 ml of the sampleaqueous solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Thereafter, the mixture was allowed to stand for30 minutes. As a result, the mixture was separated into two phases. Thelower phase (aqueous phase) was recovered, and the volume and theabsorbance at 280 nm thereof were measured. The amount of salicylalcohol extracted in the upper phase (polar organic solvent phase) wascalculated from a reduction in the absorbance at 280 nm of the lowerphase and the solution volume of the lower phase. As a result, it wasfound that by a single extraction operation, 87.7% of the startingamount of salicyl alcohol was extracted in the acetone phase.

Example 10b

[0157] This additional extraction operation to Example 10a was conductedexcept for use of 10 ml of isopropanol instead of 10 ml of acetone. As aresult, it was found that by a single extraction operation, 98.7% of thestarting amount of salicyl alcohol was extracted in the isopropanolphase.

Example 11 Extraction of Elladitannin and Polymers Thereof Example 11a

[0158] An aqueous sweet tea extract solution (SUNTENCHA; manufactured bySuntory Ltd.) was diluted two-fold with water to obtain a two-folddiluent. Glucose and sodium chloride were added and dissolved in thetwo-fold diluent to concentrations of 10% and 10%, respectively toobtain an aqueous solution. The absorbance at 280 nm of the aqueoussolution was measured. 20 ml of acetone was added to 20 ml of theaqueous solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. Thereafter, the mixture was allowed to stand for30 minutes. As a result, the mixture was separated into two phases. Thelower phase (aqueous phase) was recovered, and the volume and theabsorbance at 280 nm thereof were measured. The amount of effectiveingredients (i.e., elladitannin and polymers thereof) extracted in theupper phase (polar organic solvent phase) was calculated from areduction in the absorbance at 280 nm of the lower phase and thesolution volume of the lower phase. As a result, it was found that by asingle extraction operation, 43.1% of the effective ingredients wereextracted in the acetone phase. Moreover, the level of coloration in theacetone phase was about ⅓ that of the aqueous phase. Thus, it was foundthat by the extraction operation, the effective ingredients wereextracted and the decolorization effect was obtained.

Example 12 Extraction of Hesperidin from Fruit Juice

[0159] 10 ml of 15% sodium chloride solution was added to 10 ml ofCalifornia orange juice concentrate (five-fold concentrate), and stirredto homogeneity, thereby obtaining an aqueous solution. The amount ofhesperidin in the California orange juice concentrate used was measuredby HPLC using a column ODS, where the mobile phase was a mixture ofacetonitrile and water at 20:80, the flow rate was 0.5 ml/min, and thecolumn temperature was 40° C. The absorbance of the eluate was detectedat 280 nm. A specific method of detecting the amount of hesperidin isdescribed in Japanese Laid-Open Publication No. 8-80177. 20 ml ofacetone was added to 20 ml of the resultant aqueous solution, followedby vigorous stirring using a separating funnel for 5 minutes.Thereafter, the mixture was allowed to stand for 30 minutes. As aresult, the mixture was separated into two phases. The upper phase(polar organic solvent phase) was recovered, and the volume thereof wasmeasured. The amount of hesperidin was measured by HPLC as describedabove. As a result, by a single extraction operation, 75% of hesperidinwas extracted in the acetone phase.

Example 13 Extraction of Hesperidin from Glycosylation Reaction Solution

[0160] An aqueous solution in which 5% soluble starch (manufactured byMerck) and 0.5% hesperidin were dissolved was adjusted with hydrochloricacid to pH 9.0. Alkaline-resistant CGTase (described in JapaneseLaid-Open Publication No. 7-107972) was added to the aqueous solution toa concentration of 5 units/ml. Thereafter, an enzyme reaction wasallowed to proceed at 37° C. for 16 hours. After the end of the enzymereaction, a portion of the enzyme reaction solution was collected, andthe amounts of hesperidin and hesperidin glycosides were measured by aHPLC analysis method. In the HPLC analysis method, LiChrospher 100RP18(Merck; 4.0×250 mm) column was used, where the mobile phase was amixture of acetonitrile and water at 20:80, the flow rate was 0.5ml/min, and the column temperature was 40° C. The absorbance of theeluate was detected at 280 nm. A method of analyzing hesperidin andhesperidin glycosides is described in Japanese Laid-Open Publication No.8-80177. As a result, it was found that due to the enzyme reaction,about 80% of the hesperidin which was added at the start of the reactionwas converted into hesperidin glycosides.

[0161] Thereafter, glucose and sodium chloride were added and dissolvedin the enzyme reaction solution after the enzyme reaction toconcentration of 20% and 10%, respectively, to obtain a glucose andsodium chloride-containing enzyme reaction solution. An equal volume ofacetone was added to the glucose and sodium chloride-containing enzymereaction solution, followed by vigorous stirring using a separatingfunnel for 5 minutes. The mixture was allowed to stand for 30 minutes.As a result, the mixture was separated into two phases. The upperacetone phase was collected, and the volume thereof was measured, andthe amount of hesperidin glycosides dissolved in the acetone phase wasmeasured by HPLC as described above. As a result, about 45% of thehesperidin glycosides present in the enzyme reaction solution after theend of the enzyme reaction was extracted in the acetone phase. Further,an amount of acetone, which was half the volume of the enzyme reactionsolution, was added to the lower aqueous phase, stirred, and allowed tostand, and the upper phase was recovered. This additional extractionoperation was carried out three times. As a result, all of thehesperidin glycosides could be recovered.

Example 14 Extraction of Hydroquinone Glycoside from GlycosylationReaction Solution

[0162] An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DEis 7 to 9; manufactured by Matsutani Chemical Industry Co., Ltd.) and15% hydroquinone were dissolved, was adjusted with SN sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. After the end of the enzyme reaction, a portionof the enzyme reaction solution was collected, and the amounts ofhydroquinone and hydroquinone glycoside were measured by a HPLC analysismethod. In the HPLC analysis method, LiChrospher 100RP18 (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water,methanol and phosphoric acid at 80:19.7:0.3 (v/v), the flow rate was 0.5ml/min, and the column temperature was 40° C. The amounts ofhydroquinone and hydroquinone glycoside in the eluate were detected byultraviolet spectroscopy. The amount of product malto-oligosaccharidewas measured by a HPLC analysis method using the enzyme reactionsolution. In the HPLC analysis method, LiChrosorb NH₂ (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water andacetonitrile at 25:75, the flow rate was 1.0 ml/min, and the columntemperature was 40° C. Malto-oligosaccharide in the eluate was detectedby a RI detector. As a result, it was found that due to the enzymereaction, about 35% of the hydroquinone was converted into hydroquinoneglycoside, and the dextrin was degraded to glucose andmalto-oligosaccharides. Further, glucoamylase (manufactured by NagaseChemtex; brand name XL-4) was added to the enzyme reaction solutionafter the end of the enzyme reaction to a concentration of 8.8 units/ml,followed by incubation at 45° C. for 3 hours, thereby degrading theoligosaccharides in the enzyme reaction solution to glucose.

[0163] Thereafter, an equal volume of tetrahydrofuran was added to theenzyme reaction solution after the end of the degradation of theoligosaccharides, followed by vigorous stirring using a separatingfunnel for 5 minutes. The mixture was allowed to stand for 30 minutes.As a result, the mixture was separated into two phases. The uppertetrahydrofuran phase was recovered, and the volume thereof wasmeasured. The amount of hydroquinone glycoside dissolved in thetetrahydrofuran phase was analyzed by HPLC as described above. As aresult, about 65% of the hydroquinone glycoside present in the enzymereaction solution after the end of the enzyme reaction was extracted inthe tetrahydrofuran phase. Further, an amount of tetrahydrofuran, whichwas half the volume of the enzyme reaction solution, was added to thelower aqueous phase, stirred, and allowed to stand, and the upper phasewas recovered. This additional extraction operation was carried outthree times. As a result, all of the hydroquinone glycoside could berecovered.

Example 15 Extraction of Hydroquinone Glycoside from GlycosylationReaction Solution

[0164] An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DEis 7 to 9; manufactured by Matsutani Chemical Industry Co., Ltd.) and15% hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. After the end of the enzyme reaction, a portionof the enzyme reaction solution was collected, and the amounts ofhydroquinone and hydroquinone glycoside were measured by a HPLC analysismethod. In the HPLC analysis method, LiChrospher 100RP18 (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water,methanol and phosphoric acid at 80:19.7:0.3 (v/v), the flow rate was 0.5ml/min, and the column temperature was 40° C. The amounts ofhydroquinone and hydroquinone glycoside in the eluate were detected byultraviolet spectroscopy. The amount of product malto-oligosaccharidewas measured by a HPLC analysis method using the enzyme reactionsolution. In the HPLC analysis method, LiChrosorb NH₂ (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water andacetonitrile at 25:75, the flow rate was 1.0 ml/min, and the columntemperature was 40° C. Malto-oligosaccharide in the eluate was detectedby a RI detector. As a result, it was found that due to the enzymereaction, about 35% of the hydroquinone was converted into hydroquinoneglycoside, and the dextrin was degraded to glucose andmalto-oligosaccharides. Further, glucoamylase (manufactured by NagaseChemtex; brand name XL-4) was added to the enzyme reaction solutionafter the end of the enzyme reaction to a concentration of 8.8 units/ml,followed by incubation at 45° C. for 3 hours, thereby degrading theoligosaccharides in the enzyme reaction solution to glucose.

[0165] Thereafter, the enzyme reaction solution after the degradation ofthe oligosaccharides was vacuum concentrated by a factor of about 1.4using an evaporator. Ammonium sulfate was added to the concentrate to aconcentration of 20% to obtain an ammonium sulfate-containingconcentrate. An equal volume of acetone was added to the ammoniumsulfate-containing concentrate, followed by vigorous stirring using aseparating funnel for 5 minutes. The mixture was allowed to stand for 30minutes. As a result, the mixture was separated into two phases. Theupper acetone phase was recovered, and the volume thereof was measured.The amount of hydroquinone glycoside dissolved in the acetone phase wasanalyzed by HPLC as described above. As a result, about 60% of thehydroquinone glycoside present in the enzyme reaction solution after theend of the enzyme reaction was extracted in the acetone phase. Further,an amount of acetone, which was 0.8 times the volume of the concentrate,was added to the lower aqueous phase, stirred, and allowed to stand, andthe upper phase was recovered. This additional extraction operation wascarried out three times. As a result, all of the hydroquinone glycosidecould be recovered.

Example 16 Extraction of Hydroquinone Glycoside from GlycosylationReaction Solution

[0166] An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DEis 7 to 9; manufactured by Matsutani Chemical Industry Co., Ltd.) and15% hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. After the end of the enzyme reaction, a portionof the enzyme reaction solution was collected, and the amounts ofhydroquinone and hydroquinone glycoside were measured by a HPLC analysismethod. In the HPLC analysis method, LiChrospher 100RP18 (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water,methanol and phosphoric acid at 80:19.7:0.3 (v/v), the flow rate was 0.5ml/min, and the column temperature was 40° C. The amounts ofhydroquinone and hydroquinone glycoside in the eluate were detected byultraviolet spectroscopy. The amount of product malto-oligosaccharidewas measured by a HPLC analysis method using the enzyme reactionsolution. In the HPLC analysis method, LiChrosorb NH₂ (Merck; 4.0×250mm) column was used, where the mobile phase was a mixture of water andacetonitrile at 25:75, the flow rate was 1.0 ml/min, and the columntemperature was 40° C. Malto-oligosaccharide in the eluate was detectedby a RI detector. As a result, it was found that due to the enzymereaction, about 35% of the hydroquinone was converted into hydroquinoneglycoside, and the dextrin was degraded to glucose andmalto-oligosaccharides. Further, glucoamylase (manufactured by NagaseChemtex; brand name XL-4) was added to the enzyme reaction solutionafter the end of the enzyme reaction to a concentration of 8.8 units/ml,followed by incubation at 45° C. for 3 hours, thereby degrading theoligosaccharides in the enzyme reaction solution to glucose.

[0167] Thereafter, an equal volume of acetonitrile was added to theenzyme reaction solution after the degradation of the oligosaccharides,followed by vigorous stirring using a separating funnel for 5 minutes.The mixture was allowed to stand for 1 hour. As a result, the mixturewas separated into two phases. The upper acetonitrile phase wasrecovered, and the amount thereof was measured. The amount ofhydroquinone glycoside dissolved in the acetonitrile phase was analyzedby HPLC as described above. As a result, about 30% of the hydroquinoneglycoside present in the enzyme reaction solution after the end of theenzyme reaction was extracted in the acetonitrile phase. Further, avolume of acetonitrile, which was equal to the volume of the enzymereaction solution, was added to the lower aqueous phase, stirred, andallowed to stand, and the upper phase was recovered. This additionalextraction operation was carried out five times. As a result, about 90%of the hydroquinone glycoside which was present in the enzyme reactionsolution after the end of the enzyme reaction could be recovered.

Example 17 Study of Conditions for Glucoamylase Treatment

[0168] When a glycosylation enzyme reaction is conducted using dextrinor starch as a starting material, a large amount of glucose and dextrinhaving a lower molecular weight than that of a starting material ispresent in the enzyme reaction solution after the end of the reaction.Even if the concentration of overall saccharides is the same, the higherthe mole value, the lesser the transfer of saccharide to an organicphase. Therefore, in order to efficiently degrade dextrin remainingafter the end of the glycosylation enzyme reaction to glucoses,conditions for the degradation reaction of dextrin were studied.

[0169] An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DEis 7 to 9; manufactured by Matsutani Chemical Industry Co., Ltd.) and15% hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction.

[0170] 1.4 μl (1-fold amount), 2.1 μl (1.5-fold amount) or 2.8 μl(2-fold amount) of glucoamylase (manufactured by Nagase Chemtex; brandname XL-4) was added to 1 ml of the glycosylation reaction solutionafter the end of the reaction. Thereafter, the mixture was incubated at45° C. for 3 or 4 hours. After the incubation, the contents of glucoseand maltose in the solution were confirmed by a HPLC analysis method. Inthe HPLC analysis method, LiChrosorb NH₂ (Merck; 4.0×250 mm) column wasused, where the mobile phase was a mixture of water and acetonitrile at25:75, the flow rate was 1.0 ml/mm, and the column temperature was 40°C. Glucose and maltose in the eluate were detected by a RI detector. Theamounts of hydroquinone and hydroquinone glycoside in the eluate weredetected by a HPLC analysis method. In the HPLC analysis method,LiChrospher 100RP18 (Merck; 4.0×250 mm) column was used, where themobile phase was a mixture of water, methanol and phosphoric acid at80:19.7:0.3 (v/v), the flow rate was 0.5 ml/min, and the columntemperature was 40° C. The amounts of hydroquinone and hydroquinoneglycoside in the eluate were detected by ultraviolet spectroscopy. Basedon the resultant amounts of the obtained hydroquinone and hydroquinoneglycoside (HQG), the hydroquinone glycosylation rate was calculated. Theresults are shown in Table 8 below. TABLE 8 Glucose Maltose HQGconcentration concentration glycosylation (%) (%) rate (%) Samples 3 hrs4 hrs 3 hrs 4 hrs 3 hrs 4 hrs (XL-4) 11.6 12.2 5.2 4.9 36.1 36.2 1-fold(XL-4) 13.9 14.7 4.2 3.7 36.1 36.2 1.5-fold (XL-4) 15.2 15.8 3.0 2.636.1 36.2 2-fold

[0171] Even when saccharide degradation was performed using the 1-foldamount of glucoamylase for 3 or 4 hours, the enzyme reaction solutionhad substantially the same composition as before the addition ofglucoamylase. Therefore, it was suggested that with 1-fold glucoamylase,extension of a reaction by about one or two hours is not likely todramatically reduce the amount of maltose.

[0172] Further, even if the amount of glucoamylase was increased, theglycosylation rate was not substantially changed. This indicates thatglucoamylase can degrade dextrin without degrading hydroquinoneglycoside which is a product of interest. Therefore, it was found thatfor promotion of dextrin degradation, an increase in the amount ofglucoamylase is more effective than extension of degradation reactiontime.

[0173] Next, an influence of a difference between glucoamylasetreatments on THF treatment was studied. 1 ml of (XL-4) treatmentsolution and 1 ml of THF were mixed together, followed by vigorousshaking using a separating funnel for 1 minute. After shaking, themixture was allowed to stand for 1 hour. As a result, the mixture wasseparated into two phases. The aqueous phase was recovered. Water wasadded to the aqueous phase to 1 ml. The amounts of glucose and maltosewere measured by an HPLC analysis method using the aqueous phase. In theHPLC analysis method, LiChrosorb NH₂ (Merck; 4.0×250 mm) column wasused, where the mobile phase was a mixture of water and acetonitrile at25:75, the flow rate was 1.0 ml/min, and the column temperature was 40°C. Glucose and maltose in the eluate were detected by a RI detector. Theamounts of hydroquinone and hydroquinone glycoside in the eluate weremeasured by a HPLC analysis method. In the HPLC analysis method,LiChrospher 100RP18 (Merck; 4.0×250 mm) column was used, where themobile phase was a mixture of water, methanol and phosphoric acid at 80:19.7:0.3 (v/v), the flow rate was 0.5 ml/mm, and the column temperaturewas 40° C. The amounts of hydroquinone and hydroquinone glycoside in theeluate were measured by ultraviolet spectroscopy. Based on the obtainedresults and the above-described glucose concentration, maltoseconcentration and HQG concentration before extraction with THF, theextraction rate to the THF phase was calculated. The results are shownin Table 9 below. TABLE 9 Extraction rate to THF (%) Samples GlucoseMaltose HQG (XL-4) 43.3 38.6 71.2 1-fold (XL-4) 36.3 33.0 70.0 1.5-fold(XL-4) 38.0 36.4 73.7 2-fold

[0174] As a result, it was confirmed that as the glucose concentrationwas increased by the glucoamylase treatment, the extraction of glucoseto the THF phase tended to be suppressed.

[0175] A tendency was also confirmed, in which the larger the amount ofglucoamylase added to the sample, the smaller the volume of the aqueousphase recovered after shaking with THF and separation (the smaller theamount of water transferred to the THF phase).

Example 18 Influence of Saccharide Concentration on Extraction to aTetrahydrofuran Phase)

[0176] An influence of glucose concentration in an aqueous solution onextraction to a tetrahydrofuran (THF) phase was studied.

[0177] Initially, an aqueous solution containing 42% glucose and 9%hydroquinone glycoside was prepared. This aqueous solution was seriallydiluted with water to prepare aqueous solutions having glucoseconcentrations from 40% to 20%. Needless to say, by the dilution of theaqueous solution, not only glucose but also hydroquinone glycoside wasdiluted. 5 ml of THF was added to 5 ml of the thus-prepared aqueoussolution, followed by stirring at 30° C. for 5 minutes using a mixer.After the stirring, the mixture was centrifuged at 3000 rpm for 5minutes. The upper phase was recovered. As the glucose concentrationdecreased, the amount of the THF phase was lessened. When the glucoseconcentration was 20%, phase separation did not occur by a singleextraction operation. 5 ml of THF was added to the lower phase again andthen the upper phase was similarly recovered. The two upper phasesobtained for the aqueous solutions was added together. The contents ofglucose and maltose were measured by a HPLC analysis method. In the HPLCanalysis method, LiChrosorb NH₂ (Merck; 4.0×250 mm) column was used,where the mobile phase was a mixture of water and acetonitrile at 25:75,the flow rate was 1.0 ml/min, and the column temperature was 40° C.Glucose and maltose in the eluate were detected by a RI detector. Theamounts of hydroquinone and hydroquinone glycoside in the eluate weredetected by a HPLC analysis method. In the HPLC analysis method,LiChrospher 100RP18 (Merck; 4.0×250 mm) column was used, where themobile phase was a mixture of water, methanol and phosphoric acid at80:19.7:0.3 (v/v), the flow rate was 0.5 ml/min, and the columntemperature was 40° C. The amounts of hydroquinone and hydroquinoneglycoside in the eluate were measured by ultraviolet spectroscopy.

[0178] The results are shown in FIG. 1. As can be seen from FIG. 1, thetransfer rate of hydroquinone glycoside was not much dependent on theglucose concentration. However, the transfer rate of glucose decreasedwith an increase in the glucose concentration. Accordingly, it was foundthat if the saccharide concentration of an aqueous solution for use inextraction is increased, the transfer of a saccharide to a polar organicsolvent phase is less than the transfer of a hydrophobicgroup-containing water-soluble organic compound, whereby a high-purityhydrophobic group-containing water-soluble organic compound can beobtained.

Example 19 Influence of Concentration of Glycosylation Reaction Solutionon Extraction of Hydroquinone Glycoside

[0179] As shown in the above-described Example 18, it was found that ifthe saccharide concentration of an aqueous solution is increased, thetransfer of the saccharide to an organic phase is suppressed, so that ahydrophobic group-containing water-soluble organic compound isefficiently transferred to the organic phase. Therefore, it wasconfirmed whether or not by concentrating an enzyme reaction solutioncontaining a hydrophobic group-containing water-soluble organiccompound, the transfer of a saccharide to an organic phase wassuppressed, so that the extraction efficiency of the hydrophobicgroup-containing water-soluble organic compound was increased.

[0180] An aqueous solution, in which 35% dextrin (PINE-DEX #1 where DEis 7 to 9; manufactured by Matsutani Chemical Industry Co., Ltd.) and15% hydroquinone were dissolved, was adjusted with 5N sodium hydroxideaqueous solution to pH 6.5. A glycosylation enzyme (amylase X-23described in Japanese Laid-Open Publication No. 6-277053) was added tothe aqueous solution to a concentration of 20 units/ml. Therefore,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. 2.1 μl (1.5-fold amount) of glucoamylase(manufactured by Nagase Chemtex; brand name XL-4) was added per ml ofthe glycosylation reaction solution after the end of the reaction.Thereafter, glucoamylase treatment was performed at 45° C. for 4 hours,thereby obtaining a reaction solution.

[0181] 5 ml of this reaction solution was condensed by an evaporator tobe concentrated to 80% (4.0 ml) or 70% (3.5 ml) by volume. An equalvolume of THF was added to an unconcentrated reaction solution, the 80%concentrate or the 70% concentrate, followed by vigorous shaking using amixer for 1 minute. Extraction was conducted under a condition of 30° C.The mixture was allowed to stand for 1 hour. The mixture was separatedinto two phases. The upper THF phase was recovered. The resultant THFphase was analyzed by HPLC as described above for the contents ofglucose, maltose and hydroquinone glycoside. A step of adding an amountof THF, which was 0.5 times the amount of the aqueous phase, to thelower aqueous phase again, shaking, extraction, and recovering the upperTHF phase, was further repeated twice. The obtained THF phases werecombined together. Note that although an intermediate layer wasgenerated in the concentrate during the phase separation, theintermediate layer was included in the aqueous phase. The results areshown in the following Tables 10 to 12. TABLE 10 Unconcentrated amylasetreatment reaction solution (Brix concentration of two-fold diluent (Bx)= 24.5) Volume of Corrected concentration solution (equivalent in 5 ml)% Extraction rate (%) Samples (ml) Glucose Maltose HQG Glucose MaltoseHQG Unconcentrated 5 18.8 1.7 10.8 amylase treatment reaction solutionTHF phase total 9.5 7.4 0.1 9.9 39.2 3.2 90.9

[0182] TABLE 11 80% concentrate (two-fold diluent Bx = 29.5) (glucose22.7%, maltose: 1.3%, HQG: 13.5%) Volume of Corrected concentrationsolution (equivalent in 5 ml) % Extraction rate (%) Samples (ml) GlucoseMaltose HQG Glucose Maltose HQG GA treatment 4 18.2 1.1 10.8 solution80% concentrate THF phase total 9.2 4.7 0 10.9 26.1 0 101.1

[0183] TABLE 12 70% concentrate (two-fold diluent Bx = 34.0) (glucose25.8%, maltose: 1.9%, HQG: 15.2%) Volume of Corrected concentrationsolution (equivalent in 5 ml) % Extraction rate (%) Samples (ml) GlucoseMaltose HQG Glucose Maltose HQG GA treatment 3.5 18 1.3 10.6 GAtreatment 3.5 18 1.3 10.6 solution 70% concentrate THF phase total 7.73.9 0 10.1 21.7 0 95.2

[0184] As a result, as expected, by performing THF extraction afterconcentrating the post-glucoamylase treatment reaction solution, theamount of glucose extracted in the THF phase could be suppressed toabout 50% to 60% of that extracted from the unconcentrated solution.

Example 20 Influence of a Hydrophobic Group-Containing Water-SolubleOrganic Compound on Phase Separation

[0185] As described in Example 17, when a glycosylation reactionsolution was subjected to extraction using THF, an aqueous phase wascompletely separated from the organic phase even if the glucoseconcentration was no more than 20%, and the THF phase was larger thanthat of the aqueous phase. On the other hand, in the above-describedExample 18, when a solution containing 42% glucose and 9% hydroquinoneglycoside (containing substantially no hydroquinone) was diluted withwater to a glucose concentration of 20%, phase separation was notobtained in spite of addition of THF. Therefore, an influence of ahydrophobic group-containing water-soluble organic compound on phaseseparation was investigated.

[0186] Hydroquinone was added to a 20% glucose-containing diluent asprepared in the above-described Example 18, to a concentration of 10%,thereby obtaining an aqueous solution. An equal volume of THF was addedto the aqueous solution, followed by stirring using a mixer for 5minutes. Thereafter, the mixture was allowed to stand for 1 hour. As aresult, a THF phase was completely separated from the aqueous phase.Moreover, water got mixed in the THF phase, so that the THF phase waslarger than the aqueous phase. The THF phase was recovered. The contentof a hydroquinone glycoside in THF was measured by HPLC as describedabove. As a result, it was found that a larger amount of hydroquinoneglycoside was extracted than when hydroquinone was not added. The reasonis believed to be that by adding a substance well soluble in a THFphase, such as hydroquinone, to an aqueous phase, THF is not easilytransferred to the aqueous phase, so that separation was more efficientand a high concentration of hydroquinone was dissolved in THF so thatthe hydroquinone promoted the transfer of the glycoside.

Example 21 Influence of Temperature on Extraction to a TetrahydrofuranPhase

[0187] The influence of temperature on extraction to a tetrahydrofuranphase was investigated.

[0188] Initially, an aqueous solution, in which 35% dextrin (PINE-DEX #1where DE is 7 to 9; manufactured by Matsutani Chemical Industry Co.,Ltd.) and 15% hydroquinone were dissolved, was adjusted with 5N sodiumhydroxide aqueous solution to pH 6.5. A glycosylation enzyme (amylaseX-23 described in Japanese Laid-Open Publication No. 6-277053) was addedto the aqueous solution to a concentration of 20 units/ml. Thereafter,incubation was performed at 45° C. for 40 hours to cause a hydroquinoneglycosylation reaction. After the end of the reaction, 2.1 μl (1.5-foldamount) of glucoamylase (manufactured by Nagase Chemtex; brand nameXL-4) was added to each 1 ml of the glycosylation reaction solution.Thereafter, the mixture was incubated at 45° C. for 4 hours to obtain apost-glucoamylase treatment reaction solution.

[0189] 5 ml of THF was added to 5 ml of the post-glucoamylase treatmentreaction solution, followed by stirring using a mixer at 3, 10, 22, or45° C. for 5 minutes. Thereafter, the mixture was centrifuged at 3000rpm for 5 minutes. As a result, the mixture was separated into twophases. The aqueous phase was recovered. Water was added to the aqueousphase to a volume of 5 ml to obtain an aqueous solution. After theadjustment, the hydroquinone, hydroquinone glycoside and glucoseconcentrations of the aqueous solution were measured by HPLC asdescribed above.

[0190] The results are shown in Table 13. TABLE 13 the hydroquinone,hydroquinone glycoside and glucose concentrations of the aqueous phaseStart 3° C. 10° C. 20° C. 22° C. 30° C. 45° C. HQ 9.5 0.9 0.7 1.0 1.01.0 1.2 HQG 11.5 2.8 2.7 3.4 3.5 3.5 4.8 Glucose 19.4 11.1 11.5 13.313.4 13.4 16.6

[0191] Based on the results in Table 13, the transfer rate ofhydroquinone glycoside and glucose were calculated. FIG. 2 shows a graphof the transfer rate.

[0192] As can be seen from Table 13 and FIG. 2, when the extractiontemperature was low, a large amount of saccharide was transferred to theTHF phase. As the temperature was increased, the transfer rates ofhydroquinone, hydroquinone glycoside and glucose to the THF phase weredecreased. The reason is believed to be that the solubility of HQG andglucose to the aqueous phase increased with an increase in temperature,so that the transfer to the THF phase was decreased. Further, the higherthe temperature, the larger the difference between the transfer rates ofhydroquinone glycoside and glucose. Therefore, it was found that thepurity of hydroquinone glycoside can be more increased by performingextraction at as high a temperature as possible.

INDUSTRIAL APPLICABILITY

[0193] According to the present invention, a method of extracting ahydrophobic group-containing water-soluble organic compound is provided.The extracting method and the purifying method of the present inventioncan be used as a technique for extracting and purifying an effectiveingredient from various animals and plants, or as a technique ofextracting and purifying an effective ingredient from various enzymereaction solutions. According to the method of the present invention, ahydrophobic group-containing water-soluble organic compound can beeasily and inexpensively separated and purified.

1. A method for extracting a hydrophobic group-containing water-solubleorganic compound, comprising the step of: bringing an aqueous solutioncontaining the hydrophobic group-containing water-soluble organiccompound and a saccharide into contact with a polar organic solvent toobtain an aqueous phase and an organic phase, whereby the hydrophobicgroup-containing water-soluble organic compound is transferred to theorganic phase; wherein the hydrophobic group-containing water-solubleorganic compound is selected from the group consisting of hydroquinoneglycoside, catechin, hesperidin, hesperidin glycosides, caffeic acid,salicyl alcohol, and elladitannin; and wherein the polar organic solventis tetrahydrofuran or acetonitrile.
 2. A method according to claim 1,wherein the saccharide concentration of the aqueous solution is at least12 g per 100 ml of the aqueous solution.
 3. A method according to claim1, wherein the amount of the polar organic solvent is 0.2 to 2 times thevolume of the aqueous solution.
 4. (Cancelled)
 5. (Cancelled) 6.(Cancelled)
 7. (Cancelled)
 8. (Cancelled)
 9. (Cancelled)
 10. (Cancelled)11. A method according to claim 1, wherein the hydrophobicgroup-containing water-soluble organic compound is derived from anenzyme reaction solution.
 12. A method according to claim 11, whereinthe enzyme reaction solution is a glycosylation reaction solution.
 13. Amethod according to claim 12, wherein the glycosylation reactionsolution is a hesperidin or hydroquinone glycosylation reactionsolution.
 14. A method according to claim 1, wherein the hydrophobicgroup-containing water-soluble organic compound is derived from anorganism selected from animals or plants.
 15. A method according toclaim 1, wherein the hydrophobic group-containing water-soluble organiccompound is derived from fruit juice.
 16. A method according to claim 1,wherein the aqueous solution is prepared by concentrating an enzymereaction solution containing the hydrophobic group-containingwater-soluble organic compound and the saccharide.
 17. A methodaccording to claim 16, wherein the enzyme reaction solution is aglycosylation reaction solution.
 18. A method according to claim 17,wherein the glycosylation reaction solution is a hesperidin orhydroquinone glycosylation reaction solution.
 19. A method according toclaim 1, wherein the aqueous solution is prepared by concentrating ordiluting an extract of an organism, wherein the extract contains thehydrophobic group-containing water-soluble organic compound and thesaccharide, and the organism is an animal or a plant.
 20. A methodaccording to claim 1, wherein the aqueous solution is prepared byconcentrating fruit juice.
 21. (Cancelled)
 22. (Cancelled) 23.(Cancelled)
 24. (Cancelled)
 25. (Cancelled)
 26. A method for purifying aphenol derivative glycoside, comprising the steps of: bringing a firstaqueous solution containing a phenol derivative, a phenol derivativeglycoside and a saccharide into contact with a polar organic solvent toobtain a first aqueous phase and an organic phase containing a smallamount of water, whereby the phenol derivative and the phenol derivativeglycoside are transferred to the organic phase; recovering the organicphase containing the small amount of water; removing the polar organicsolvent from the organic phase containing the small amount of water toobtain a second aqueous solution containing the phenol derivative andthe phenol derivative glycoside; bringing the second aqueous solutioninto contact with ethyl acetate to obtain a second aqueous phase and anethyl acetate phase, whereby the phenol derivative is transferred to theethyl acetate phase; recovering the second aqueous phase; andconcentrating and cooling the second aqueous phase to precipitate thephenol derivative glycoside; wherein the phenol derivative and thephenol derivative glycoside are derived from a phenol derivativeglycosylation reaction solution; wherein the glycosylation reactionsolution is a hesperidin or hydroquinone glycosylation reactionsolution; and wherein the polar organic solvent is tetrahydrofuran oracetonitrile.
 27. A method according to claim 26, wherein the amount ofthe polar organic solvent is 0.2 to 2 times the volume of the firstaqueous solution.
 28. A method according to claim 26, wherein theglycosylation reaction solution is a hydroquinone glycosylation reactionsolution.
 29. (Cancelled)
 30. (Cancelled)